In Project 4 during the previous funding period, specific aims 2 and 3- led to the discovery of an Invasion Signature in rat and mouse mammary tumors. As a result, significant effort was expended to define the particular relevance of the Invasion Signature to the chemotactic migratory behavior of metastatic cancer cells during invasion and intravasation in mouse mammary tumors. We found that the genes coding for pathways leading to the ZBP1/mRNA targeting, cofilin, Mena/capping protein and N-WASP/Arp2/3 pathways, that regulate EMT and beta-actin polymerization during invasion, and the directionality of cell protrusion and contractility during chemotaxis to EGF, are coordinately regulated. Coordinate regulation of these genes is particularly relevant to the contribution of the tumor microenvironment to metastasis because chemotaxis to blood vessels is involved in the escape of cancer cells from primary mammary tumors. The ZBP1, cofilin, Mena and N-WASP pathways, emanating from PI3K, were studied in Project 4, with the help of all Cores, for their ability to alter metastatic outcome and the results confirmed the importance of the Invasion Signature in metastasis in mammary tumors. This confirmation stimulated detailed analysis of these pathways in chemotaxis and invasion of tumor cells and these studies have generated new insights into the molecular mechanisms of chemotaxis during metastasis. The Invasion Signature also contains a metastasis suppressor pathway, the ZBP1/mRNA targeting pathway, which contributes to cadheren junction stability and suppression of EMT. A major insight to emerge from these studies was that PI3K is the starting point from which these pathways emanate indicating its critical importance in tumor cell migration leading to the current Project 4 of this competing renewal. Project 5 also evolved from this work. The theme of Project 5 is to extend the new technologies and findings of the previous funding period in rats and mice to human breast tumors to determine if microenvironments exist in human tumors similar to those discovered in rats and mice during the previous funding period and if they can be used as landmarks in correlation with biomarkers to predict outcome. Combining multiphoton defined landmarks and molecular pathology studies with biomarkers derived from the Invasion Signature of the previous funding period, the prognostic value of master genes will be determined for human breast cancer. In addition, we are interested in determining if an invasion signature exists in humans, that is similar to that in rats and mice, containing master genes that can be used as biomarkers to predict outcome in human patients.